Pertussis (also known as whooping cough) continues to be a global health problem with an estimated 45 million cases annually and 300,00 deaths, which occur mostly in children. Despite high vaccine coverage, incidence has been increasing in several developed countries since the 1990s, due apparently to waning immunity in adolescents and young adults who were vaccinated, rather than infected, as children, especially those vaccinated with acellular vaccines. Understanding how vaccine components function in pathogenesis and immunity is critical to the development of improved vaccines. The causative agents of pertussis are Bordetella pertussis and Bordetella parapertussishu. These strictly human-specific pathogens are extremely closely related to Bordetella bronchiseptica, which has a broad host range that includes nearly all mammals. We have been taking a comparative approach, including all three of these Bordetella subspecies to study the roles of virulence gene regulation and of specific virulence factors in pathogenesis. Using B. bronchiseptica allows us to study respiratory infection in the lab in the context of a natural bacterial-host interaction, and the use of chimeric B. bronchiseptica and B. pertussis strains that express heterologous virulence factors has allowed us to demonstrate that several virulence factors, including Filamentous hemagglutinin (FHA), are functionally interchangeable between these subspecies. FHA is a large surface-associated and secreted protein and one of the primary components of pertussis vaccines. We have shown that FHA is required for colonization of the lower respiratory tract (specifically the trachea) and that it allows Bordetella to suppress the inflammatory response of its hosts. FHA is also one of the prototypical members of the Two Partner Secretion (TPS) pathway family. Our previous studies showed that the topology of FHA on the bacterial surface was the opposite of what had been assumed and led to the development of a new model for TPS. Our studies also demonstrated the importance of studying secretion and function simultaneously as results from secretion experiments revealed insight into function and vice versa. We propose experiments to determine how FHA modulates inflammation in the respiratory tract, to identify host cell receptors for FHA, and to identify specific regions and amino acids within FHA that are important for receptor binding. In addition to revealing mechanisms of FHA function, our results may identify novel pathways by which inflammation is controlled in general. We will also determine how the C-terminus of the FHA precursor, FhaB, controls maturation and release of FHA from the bacterial surface. Our results will provide insight into the TPS mechanism, and may set a new paradigm for how proteins are folded on cell surfaces.